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1.
Blood ; 130(19): 2047-2049, 2017 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-29122770
2.
Arterioscler Thromb Vasc Biol ; 33(2): 275-84, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23202369

RESUMEN

OBJECTIVE: The principle role of the vascular endothelium is to present a semi-impermeable barrier to soluble factors and circulating cells, while still permitting the passage of leukocytes from the bloodstream into the tissue. The process of diapedesis involves the selective disruption of endothelial cell junctions, which could compromise vascular integrity. It is therefore somewhat surprising that neutrophil transmigration does not significantly impair endothelial barrier function. We examined whether neutrophils might secrete factors that promote vascular integrity during the latter stages of neutrophil transmigration, in particular, the role of neutrophil serine proteinase 3 (PR3). METHODS AND RESULTS: Endothelial cells were treated with PR3 either in its soluble form or in a complex form with cell surface NB1. We observed that PR3 mediated the enhancement of endothelial cell junctional integrity and that this required its proteolytic activity, as well as endothelial cell expression of the protease-activated receptor-2. Importantly, PR3 suppressed the vascular permeability changes and disruption of junctional proteins induced by the action of protease-activated receptor-1 agonists. CONCLUSIONS: These findings establish the potential for neutrophil-derived PR3 to play a role in reestablishing vascular integrity after leukocyte transmigration and in protecting endothelial cells from protease-activated receptor-1-induced permeability changes that occur during thrombotic and inflammatory events.


Asunto(s)
Permeabilidad Capilar , Células Endoteliales de la Vena Umbilical Humana/enzimología , Mieloblastina/metabolismo , Neutrófilos/enzimología , Receptor PAR-2/metabolismo , Migración Transendotelial y Transepitelial , Señalización del Calcio , Técnicas de Cocultivo , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , Isoantígenos/metabolismo , Mieloblastina/genética , Interferencia de ARN , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Tiempo , Transfección , Proteína de Unión al GTP rac1/metabolismo
3.
J Immunol ; 188(5): 2419-26, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22266279

RESUMEN

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1ß-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.


Asunto(s)
Isoantígenos/biosíntesis , Mieloblastina/fisiología , Neutrófilos/enzimología , Neutrófilos/inmunología , Receptores de Superficie Celular/biosíntesis , Migración Transendotelial y Transepitelial/inmunología , Activación Enzimática/genética , Activación Enzimática/inmunología , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/fisiología , Regulación de la Expresión Génica/inmunología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Isoantígenos/genética , Isoantígenos/fisiología , Mieloblastina/biosíntesis , Mieloblastina/genética , Neutrófilos/citología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología
4.
Thromb Haemost ; 99(2): 363-72, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18278187

RESUMEN

A common feature of severe sepsis is vascular inflammation and damage to the endothelium. Because platelets can be directly activated by bacteria and endotoxin, these cells may play an important role in determining the outcome of sepsis. For example, inhibiting platelet interactions with the endothelium has been shown to attenuate endothelial cell damage and improve survival during sepsis. Although not entirely understood, the interactions between bacteria-activated platelets and the endothelium may play a key role in the vascular pathology of bacterial sepsis. Haemophilus somnus is a bacterial pathogen that causes diffuse vascular inflammation and endothelial damage. In some cases H. somnus infection results in an acute and fatal form of vasculitis in the cerebral microvasculature known as thrombotic meningoencephalitis (TME). In this study, we have characterized the mechanisms involved in endothelial cell apoptosis induced by activated platelets. We observed that direct contact between H. somnus-activated platelets and endothelial cells induced significant levels of apoptosis; however, Fas receptor activation on bovine endothelial cells was not able to induce apoptosis unless protein synthesis was disrupted. Endothelial cell apoptosis by H. somnus-activated platelets required activation of both caspase-8 and caspase-9, as inhibitors of either caspase inhibited apoptosis. Furthermore, activated platelets induced endothelial cell production of reactive oxygen species (ROS) and disrupting ROS activity in endothelial cells significantly inhibited apoptosis. These findings suggest that bacterial activation of platelets may contribute to endothelial cell dysfunction observed during sepsis, specifically by inducing endothelial cell apoptosis.


Asunto(s)
Apoptosis , Plaquetas/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Células Endoteliales/metabolismo , Haemophilus somnus/patogenicidad , Activación Plaquetaria , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Plaquetas/virología , Caspasa 3/metabolismo , Inhibidores de Caspasas , Bovinos , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/patología , Activación Enzimática , Proteína Ligando Fas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo
5.
Shock ; 29(2): 189-96, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18386389

RESUMEN

Histophilus somni is a gram-negative coccobacillus that causes respiratory and reproductive disease in cattle. The hallmark of systemic H. somni infection is diffuse vascular inflammation that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis. Previously, we demonstrated that H. somni and its lipooligosaccharide (LOS) activate bovine platelets, leading to expression of P selectin, CD40L, and FasL. Because activated platelets have been reported to induce endothelial cell cytokine production and adhesion molecule expression, we sought to determine if bovine platelets induce proinflammatory and procoagulative changes in bovine pulmonary artery endothelial cells. Endothelial cells were incubated with platelets activated with adenosine diphosphate, H. somni, or H. somni LOS. Incubation with activated bovine platelets significantly increased expression of in adhesion molecules (intercellular adhesion molecule 1, E selectin) and tissue factor, as measured by flow cytometry, real-time polymerase chain reaction, and Western blot analysis. Activated platelets also up-regulated expression of endothelial cell IL-1beta, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1alpha as determined by real-time polymerase chain reaction and an IL-1beta enzyme-linked immunosorbent assay. An interesting and surprising finding was that bovine platelets activated by H. somni or its LOS were internalized by bovine endothelial cells as visualized by transmission electron microscopy. This internalization seemed to correlate with endothelial cell activation and morphological changes indicative of cell stress. These findings suggest that activated platelets might play a role in promoting vascular inflammation during H. somni infection.


Asunto(s)
Plaquetas/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Haemophilus somnus/metabolismo , Activación Plaquetaria/fisiología , Animales , Plaquetas/metabolismo , Plaquetas/ultraestructura , Western Blotting , Bovinos , Células Cultivadas , Quimiocina CCL2/metabolismo , Selectina E/genética , Selectina E/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Haemophilus somnus/fisiología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Microscopía Electrónica de Transmisión , Activación Plaquetaria/efectos de los fármacos , Polisacáridos Bacterianos/farmacología , Arteria Pulmonar/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
6.
J Vet Diagn Invest ; 15(1): 72-6, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12580302

RESUMEN

Concerns about retroviruses in livestock and products derived from them have necessitated the development of tests to detect the bovine leukemia virus (BLV) in blood and milk from cattle. Dairy cattle (n = 101) from 5 different geographical areas were used for this study. A nested polymerase chain reaction (PCR) identified 98% of BLV seropositive cattle (n = 80) from blood and 65% from milk, whereas real-time PCR detected 94% of BLV seropositive cattle from blood and 59% from milk. Bovine leukemia virus was also detected by PCR in approximately 10% of seronegative cattle (n = 21), most likely because of early detection before seroconversion.


Asunto(s)
Sangre/virología , Leucosis Bovina Enzoótica/diagnóstico , Leucosis Bovina Enzoótica/virología , Virus de la Leucemia Bovina/genética , Virus de la Leucemia Bovina/aislamiento & purificación , Leche/virología , Reacción en Cadena de la Polimerasa/métodos , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Genes Virales , Sensibilidad y Especificidad
7.
Cardiovasc Res ; 91(1): 134-41, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21285294

RESUMEN

AIMS: Cells of the monocyte lineage are the most abundant inflammatory cells found in atherosclerotic lesions. Dominance of the inflammatory infiltrate by monocytes indicates that there is a disease-driven mechanism supporting their selective recruitment. Previous studies have demonstrated that interactions between endothelial cells (ECs) and platelets may promote monocyte recruitment. In this study, we sought to expand on this knowledge using a complex coculture model of the diseased vessel wall. METHODS AND RESULTS: Using primary human cells in an in vitro flow-based adhesion assay, we found that secretory arterial smooth muscle cells (SMCs), cocultured with ECs, promote preferential recruitment of monocytes from blood in a TGF-ß1-dependent manner. Approximately 85% of leucocytes recruited to the endothelium were CD14(+). Formation of adhesive platelet bridges on ECs was essential for monocyte recruitment as platelet removal or inhibition of adhesion to the ECs abolished monocyte recruitment. Monocytes were recruited from flow by platelet P-selectin and activated by EC-derived CC chemokine ligand 2 (CCL2), although the presentation of CCL2 to adherent monocytes was dependent upon platelet activation and release of CXC chemokine ligand 4 (CXCL4). In an intravital model of TGF-ß1-driven vascular inflammation in mice, platelets were also necessary for efficient leucocyte recruitment to vessels of the microcirculation in the cremaster muscle. CONCLUSIONS: In this study, we have demonstrated that stromal cells found within the diseased artery wall may promote the preferential recruitment of monocytes and this is achieved by establishing a cascade of interactions between SMCs, ECs, platelets, and monocytes.


Asunto(s)
Aterosclerosis/inmunología , Plaquetas/inmunología , Adhesión Celular , Células Endoteliales/inmunología , Monocitos/inmunología , Miocitos del Músculo Liso/inmunología , Activación Plaquetaria , Animales , Aterosclerosis/sangre , Plaquetas/metabolismo , Comunicación Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Masculino , Ratones , Monocitos/metabolismo , Miocitos del Músculo Liso/metabolismo , Selectina-P/metabolismo , Adhesividad Plaquetaria , Factor Plaquetario 4/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo
8.
Infect Immun ; 75(2): 1045-9, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17118985

RESUMEN

Histophilus somni-induced platelet aggregation was inhibited by antagonists of the platelet-activating factor (PAF) receptor but not inhibitors of PAF synthesis. In addition, H. somni cells expressing phosphorylcholine (ChoP) induced aggregation, while ChoP(-) H. somni cells did not. This suggests that H. somni ChoP may induce platelet aggregation via interactions with the PAF receptor.


Asunto(s)
Pasteurellaceae/fisiología , Fosforilcolina/metabolismo , Agregación Plaquetaria , Animales , Adhesión Bacteriana , Plaquetas/microbiología , Bovinos , Pasteurellaceae/genética , Factor de Activación Plaquetaria/antagonistas & inhibidores , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal
9.
Microb Pathog ; 38(1): 23-32, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15652292

RESUMEN

Haemophilus somnus is a bacterial pathogen that causes respiratory disease and vasculitis in cattle. Thrombotic meningoencephalitis (TME) and other severe forms of H. somnus-mediated vascular disease are characterized histopathologically by vasculitis, thrombosis, and infiltration of polymorphonuclear cells. It has been reported previously that activated human platelets express CD40L, FasL and P-selectin (CD62P). We hypothesized that if these surface markers are up-regulated on bovine platelets after in vitro exposure to H. somnus and its lipooligosaccharide (LOS), they might contribute to endothelial cell damage. Using flow cytometry, we demonstrated low baseline expression of these molecules by bovine platelets and increased expression following in vitro stimulation with ADP, H. somnus or H. somnus LOS. H. somnus stimulated platelets were capable of causing apoptosis in endothelial cells as measured by Hoechst-33342 staining and caspase-3 activity. If these events occur in vivo, they might promote vascular damage and endothelial cell apoptosis, leading to the development of vasculitis and thrombosis that characterize bovine H. somnus infection.


Asunto(s)
Apoptosis , Plaquetas/fisiología , Células Endoteliales/fisiología , Haemophilus somnus/patogenicidad , Lipopolisacáridos/toxicidad , Animales , Bencimidazoles/metabolismo , Plaquetas/química , Ligando de CD40/análisis , Caspasa 3 , Caspasas/análisis , Bovinos , Células Cultivadas , Células Endoteliales/citología , Proteína Ligando Fas , Haemophilus somnus/química , Lipopolisacáridos/aislamiento & purificación , Glicoproteínas de Membrana/análisis , Selectina-P/análisis , Activación Plaquetaria , Agregación Plaquetaria
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